CN106011157A

CN106011157A - Capsella bursa-pastoris peroxidase gene and application of capsella bursa-pastoris peroxidase gene to improvement of cold resistance of economic plants

Info

Publication number: CN106011157A
Application number: CN201610555165.4A
Authority: CN
Inventors: 林娟; 李伟伟; 陈虎; 魏东晖
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2016-07-15
Filing date: 2016-07-15
Publication date: 2016-10-12

Abstract

The invention belongs to the technical field of plant molecular biology, in particular to a capsella bursa-pastoris peroxidase gene and application of capsella bursa-pastoris peroxidase gene to improvement of cold resistance of economic plants. The nucleotide sequence of the capsella bursa-pastoris peroxidase functional gene is shown in SEQ ID NO: 1, and the capsella bursa-pastoris peroxidase gene is encoded by the nucleotide sequence, is expressed through low temperature induction, is under-expressed at normal temperature, and further promotes cells to generate related low-temperature induced genes through regulation of active oxygen in plant cells after expression, so as to finally improve the cold resistance of the plants. An over-expressed vector constructed by utilizing the gene segment can be over-expressed in the plants, and then cold resistant gene expression can be regulated and controlled, so as to protect the plant cells from being destroyed by low temperature. The invention further provides an application method for cultivating the cold resistant plants based on the gene, and the application method is used for improvement of varieties of crops with economic value.

Description

Herba Capsellae peroxidase gene and the application in improvement economic plants winter resistance thereof

Technical field

The invention belongs to molecular biology of plants technical field, be specifically related to a kind of Herba Capsellae peroxidase gene, its system Preparation Method, the recombiant plasmid of this gene, and it is applied to the regulation and control of plant adverse circumstance especially for economic plants Cold resistant genes Genetic transformation, to improve plant cold resistance.

Background technology

Temperature is indispensable a kind of environmental condition in growing process, and plant is as a kind of life of fixed growth Thing, it is impossible to hide poor environment by walking is mobile, be more exposed to the restriction of environmental factors.Low temperature freezing-disaster is tight in agricultural production The natural disaster of weight, not only can limit the plantation scope of crops, will also result in crop production reduction, the crops caused every year Lose huge, so about the research of plant cold resistance, the inherited character of transformation crops, being allowed to free from freeze injury, It always is one of focus that agriculture field studies, is also ideal and the pursuit of scientist.Development along with biotechnology With extensively application, constantly deepening the understanding of cold-resistant molecule mechanism, using modern biotechnology to cultivate cold-resistant new varieties becomes One of Main Means.

Utilizing modern molecular biology technique, people have cloned numerous participation plant low temperature tolerance ability from various plants The gene formed, these genes are all a kind of specific genes that plant induces generation at low ambient temperatures, referred to as cold-regulated genes Or Cold exposed gene, protein produced by them can protect the plants from injury from low temperature, therefore study low-temperature induction of genes Effect and be applied to crops improvement have great significance.Found in the research of model plant arabidopsis in recent years The one low-abundance low-temperature induction of genes of class, referred to as RCI(Rare Cold Inducible) gene.The RCI gene of arabidopsis is divided into Being four classes, wherein the 3rd class is RCI3 gene, and this gene encoding production is a kind of Cationic peroxidase (Llorente F, L ópez-Cobollo RM, Catalá R, Martínez-Zapater JM, Salinas J. A novel cold- inducible gene from Arabidopsis, RCI3, encodes a peroxidase that constitutes A component for stress tolerance. Plant J.2002,32 (1): 13-24), acting as in plant Can strengthen under low temperature stress the function of detoxification of active oxygen in plant cell (Reactive Oxygen Species, ROS) from And improve winter resistance (Kim MJ, Ciani S, the Schachtman DP. A peroxidase contributes of plant to ROS production during Arabidopsis root response to potassium deficiency. Mol. Plant. 2010,3 (2): 420-427).Substantial amounts of research shows when the external condition of plant occurs drastically to change, As during low temperature stress, internal to O₂Utilization ability reduce, unnecessary O₂In metabolic process, then it is converted to ROS, mistake Plant cell can be caused damage by many ROS accumulation, there is a kind of antioxidant defense mechanisms to eliminate the unfavorable work of ROS in plant With.ROS is also important Signal Regulation molecule simultaneously, participates in the growth promoter of regulation and control plant and various Stress responses.At present Research find that the generation of ROS can affect the expression of several genes in plant, illustrate plant cell during evolution Obtain the mechanism utilizing ROS as signaling molecule.ROS can be selectively applied to one as signaling molecule can perception The target protein of ROS, then hands on this signal thus regulates the change of some gene expression.In plant cell, The result of ROS regulation and control has front, also has negative, and this two-sided regulating and controlling effect of ROS obtains in various abiotic stress To confirming, therefore, ROS is produced and in purge mechanism, the functional study of various components will assist in more preferable control in cell The concentration of ROS.In plant cell, the regulation and control of the level of ROS have a good application prospect for the resistance improving crop.

Herba Capsellae (Capsella bursa-pastoris) it is a kind of 1 or 2 year wild herbaceous plant, ground of tiling, Happiness the moon, is the most visible a kind of edible vegetable in south, belongs to Cruciferae (Cruciferae), Herba Capsellae genus (Capsella), can grow and blossom and bear fruit by normal growth in the most such as early spring and autumn and winter season.Institute of the present invention The Herba Capsellae peroxidase gene related to is to clone to obtain from the DNA of the genome of Herba Capsellae blade.Not yet it is found to have at present Close Herba Capsellae peroxidase gene report in cultivating hardy plant.

Summary of the invention

The first object of the present invention is just to provide a kind of new Herba Capsellae peroxidase gene, and this gene is the base from Herba Capsellae Because in group, clone obtains, the protein of the high expressed that to be plant produce under cold-induced and Cold exposed.

The second object of the present invention is to provide a kind of new Herba Capsellae peroxidase.

The third object of the present invention is to provide described Herba Capsellae peroxidase and gene order is utilizing transgenic technology Application in improvement plant stress-resistance (low temperature resistant).

In one aspect of the invention, it is provided that a kind of isolated DNA molecule, a kind of new Herba Capsellae peroxide Enzyme gene, this gene is to clone to obtain from the genome of Herba Capsellae.This Herba Capsellae peroxidase gene, is selected from:

(1) its nucleotide sequence is as shown in 20-1532 bit base in SEQ ID No.1；

(2) from nucleotide 20-232 in its nucleotide sequence and SEQ ID No. l；337-525；615-777；1116-1532 The nucleotides sequence of position DNA molecular shows at least 70% homology and coding identical function protein；Or

(3) its nucleotides sequence is listed under medium stringency conditions and nucleotide from nucleotide 20-1532 position in SEQ ID No.1 Sequence hybridization and coding identical function protein.

In another aspect of this invention, also providing for a kind of recombiant plasmid, it comprises above-mentioned Herba Capsellae peroxidase gene.

In another aspect of this invention, a kind of Herba Capsellae peroxidase is also provided for, comprising: have SEQ ID No.2 ammonia The polypeptide of base acid sequence or its conservative variation's polypeptide or its active fragment, or its reactive derivative.It is preferred that described Herba Capsellae Peroxidase is the polypeptide of SEQ ID No.2 sequence.

In a still further aspect thereof, the method for preparing above-mentioned Herba Capsellae peroxidase gene, institute are additionally provided The method of stating comprises the following steps:

(1) by Herba Capsellae seed after 70% alcohol disinfecting, it is seeded in MS culture medium；

(2) after described Herba Capsellae seed grows blade, extract the genomic DNA of described blade, divide with 1% agarose gel electrophoresis Analyse its quality；And

(3) genomic walking technology clone is used to obtain Herba Capsellae peroxidase gene full-length genome sequence, wherein, use Forward primer is that CbRCIF1:5-ATGAACTGCTTGAGAGCTATTGCCC-3(is designated as SEQ ID No.3), downstream primer is CbRCIR:5-TTAACTATTTGCAACGGAACATTGCCT-3(is designated as SEQ ID No.4).

In a still further aspect thereof, additionally providing the method for preparing above-mentioned recombiant plasmid, described method includes Following steps:

(1) design primer pair, amplifies the intact DNA fragments of above-mentioned Herba Capsellae peroxidase gene, the upstream of described primer pair Primer is CbRCIF1:5-ATGAACTGCTTGAGAGCTATTGCCC-3(SEQ ID No.3), described downstream primer is CbRCIR:5-TTAACTATTTGCAACGGAACATTGCCT-3(SEQ ID No.4)；And

(2) described intact DNA fragments is cloned into intermediate carrier pMD18-T, is further cloned into plant expression vector p1304 On, obtain above-mentioned recombiant plasmid.

In step (1), restriction enzyme site is introduced to described forward primerNcoI, and to described downstream primer Introduce restriction enzyme siteBstEII。

In a still further aspect thereof, additionally provide above-mentioned Herba Capsellae peroxidase gene or above-mentioned recombiant plasmid is being planted Application in thing winter resistance and tolerance to cold.It is preferred that described plant is the crop with economic worth, more preferably include Oryza sativa L., Semen Tritici aestivi, Semen Maydis, Cotton Gossypii, Brassica campestris L, Fructus Lycopersici esculenti and Fructus Cucumidis sativi.

In another aspect of this invention, a kind of host cell with above-mentioned vector is additionally provided.This place in instances Chief cell is arabidopsis, Nicotiana tabacum L. and other plant cell etc..

In another aspect of this invention, the side that a kind of generation has the polypeptide of Herba Capsellae peroxidase activity is additionally provided Method, its step is as follows:

(1) nucleotide sequence of the purification that coding has Herba Capsellae peroxidase activity polypeptide is operably coupled to expression regulation Sequence, is formed in Herba Capsellae peroxidase gene expression vector, described nucleotide sequence and SEQ ID No.1 from nucleotide The nucleotides sequence of 20-1532 position shows the homology of at least 70%；

(2) expression vector in step (1) is proceeded to prokaryotic host cell, form the reconstitution cell of Herba Capsellae peroxidase；

(3) under conditions of applicable expression Herba Capsellae peroxide enzyme polypeptide, the reconstitution cell in incubation step (2)；

(4) isolate there is the purest polypeptide of Herba Capsellae peroxidase activity.

It is preferred that the nucleotide sequence used in the method is the sequence of 20-1532 position in SEQ ID No.l.

In another aspect of this invention, additionally providing one utilizes transgenic technology that coding has Herba Capsellae peroxidase The nucleotide sequence of active polypeptide is transformed into plant to improve the plant method to the toleration of low temperature, and its step is as follows:

(1) nucleotide sequence of the purification that coding has Herba Capsellae peroxidase activity polypeptide is operably coupled to plant and expresses After regulating and controlling sequence, form the plant expression vector containing Herba Capsellae peroxidase gene, described nucleotide sequence and SEQ ID In No.1, nucleotides sequence from nucleotide 20-1532 position shows the homology of at least 70%；

(2) expression vector in step (1) is proceeded to Agrobacterium, the Agrobacterium containing expression vector is trained altogether with eukaryotic host cell Support, under the conditions of 22-28 DEG C, after light culture 1-2d, by screening such as antibiotic-screening, it is thus achieved that containing Herba Capsellae peroxidase The conversion cell of gene finally regeneration of transgenic plant and offspring thereof, including plant seed and plant tissue.Containing Herba Capsellae mistake The transfer-gen plant of oxide enzyme gene has the effect of enhancing to plant drought tolerance characteristic.

It is preferred that the nucleotides sequence used in the method is classified as the sequence of 20-1532 position in SEQ ID No.1.

In another aspect of the present invention, additionally provide the antibody being combined with Herba Capsellae peroxidase polypeptid specificity, it Including polyclonal antibody and monoclonal antibody.

In the present invention, " separation ", " purification " or " the purest " DNA refers to, this DNA or fragment are own from natural shape Be positioned in the sequence of its both sides under state and separate, also refer to this DNA. or fragment with the group of adjoint kernel thuja acid under native state Part separately, and oneself is through separating with protein with it in cell.

In the present invention, term " Herba Capsellae peroxidase (or polypeptide) gene order " refers to include coding Herba Capsellae peroxidating The nucleotide sequence of thing peptide activity, such as 20-232 in SEQ ID No.1；337-525；615-777；1116-1532 position Nucleotide sequence and degenerate sequence thereof.This degenerate sequence refers to, is positioned at the encoder block 20-1532 position of SEQ ID No.1 sequence In nucleotide, the sequence that the degenerate codon having one or more codon to be encoded same amino acid produces after being replaced. Due to the degeneracy of codon, thus with 20-232 in SEQ ID No.1；337-525；615-777；1116-1532 position is compiled The degenerate sequence of the nucleotide sequence homology the most about 70% in code district also can encode out the sequence described in SEQ ID No.2.Should Term also include can under medium stringency conditions, more preferably under high stringency conditions with SEQ ID No.1 in from nucleotide The nucleotide sequence of the nucleotide sequence hybridization of 20-1532 position.This term also include with SEQ ID No.1 in from nucleotide The homology at least 70% of the nucleotide sequence of 20-1532 position, preferably at least 80%, more preferably at least 90%, the most at least The nucleotide sequence of 95%.

This term also includes encoding the SEQ ID with the albumen with natural Herba Capsellae peroxidase identical function No. the variant form of coding region sequence in 1.These variant forms include (but being not limited to): several (usually 1-90, Preferably 1-60, more preferably 1-20, most preferably 1-10) disappearance of nucleotide, insert and/or replace, and 5 ' and/ Or 3 ' end add several (usually within 60, within preferably 30, within being more preferably 10, be most preferably 5 with In) nucleotide.

In the present invention, " the purest " protein or polypeptide refer to that it at least accounts at least the 20% of the total material of sample, preferably Ground at least 50%, more preferably at least 80%, the most at least 90%(is based on dry weight or weight in wet base).Purity can be with any suitable side Method measures, as measured the purity of polypeptide by column chromatography, PAGE or HPLC method.The purest polypeptide is substantially free of sky So with its component under state.

In the present invention, term " Herba Capsellae peroxidase or polypeptide " refers to the SEQ ID with Herba Capsellae peroxidase activity No. the polypeptide of 1 sequence.This term also includes having the SEQ ID No.2 sequence with natural Herba Capsellae peroxidase identical function Variant form.These variant forms include (but being not limited to): several (usually 1-50, preferably 1-30, more preferably Ground 1-20, most preferably 1-10) amino acid whose disappearance, insert and/or replace, and add one at C-terminal and/or N-terminal Individual or several (usually 20 within, within preferably 10, within being more preferably 5) aminoacid.Such as, in this area In, when replacing with similar nature or similar aminoacid, generally will not change the function of protein.The most such as, at C-terminal And/or N-terminal interpolation one or several aminoacid generally also will not change the function of protein.This term also includes Herba Capsellae peroxide The active fragment of compound enzyme and reactive derivative.

The variant form of the Herba Capsellae peroxide enzyme polypeptide of the present invention includes: homologous sequence, conservative variant, equipotential become Allosome, natural mutation, induced mutants, under high or low stringent condition can with Herba Capsellae peroxidase dna hybridization DNA Coded albumen and utilize the polypeptide or albumen that the serum of Herba Capsellae peroxide enzyme polypeptide obtains.

In the present invention, " Herba Capsellae peroxidase conservative variation's polypeptide " refers to and the aminoacid sequence of SEQ ID No.1 Comparing, have at most 10, the most at most 8, the aminoacid that the most at most 5 amino acid nature are similar or close is replaced And form polypeptide.These conservative variation's polypeptide are replaced preferably based on table 1 and produce.

Invention also includes the analog of Herba Capsellae peroxidase or polypeptide.These analog and natural Cold-inducible protein gene polypeptide Difference can be the difference on aminoacid sequence, it is also possible to be not affect the difference on the modified forms of sequence, or hold concurrently and There is it.These polypeptide include the natural or genetic variant of induction.Induction variant can be obtained by various technology, as passed through Radiate or be exposed to mutagenic agent and produce random mutagenesis, also by site-directed mutagenesis or the biological skill of other known moleculars Art.Analog also includes the analog with the residue (such as D-aminoacid) being different from natural L-amino acids, and has non-sky The analog of that so exist or synthesis aminoacid (such as β, gamma-amino acid).Should be understood that the polypeptide of the present invention is not limited to above-mentioned The representational polypeptide enumerated.

(the most the not changing primary structure) form of modification includes: the chemically derived form such as acetyl of inner or in vitro polypeptide Change or carboxylated.Modify and also include glycosylation, as those or are processed further in step carrying out in the synthesis of polypeptide and processing Glycosylation modified and the polypeptide that produces.This modification can carry out glycosylated enzyme (such as mammal by being exposed to by polypeptide Glycosylase or deglycosylating enzyme) and complete.Modified forms also includes having phosphorylated amino acid residue (such as phosphoric acid cheese ammonia Acid, phosphoserine, phosphothreonine) sequence.Also include being modified thus improve its Proteolytic enzyme performance or optimize The polypeptide of solubility property.

In the present invention, can be selected for various carrier known in the art, such as commercially available carrier, including plasmid, cosmid etc..? When producing the Herba Capsellae peroxide enzyme polypeptide of the present invention, can be operably coupled to Herba Capsellae peroxidase coding sequence express Regulating and controlling sequence, thus form Herba Capsellae peroxidase expression vector.

Table 1

As used in the present invention, " being operably coupled to " refers to that some part of such a situation, i.e. linear DNA molecule can affect The activity of same other parts of linear DNA molecule.Such as, if signal peptide DNA is as precursor expression the secretion that participates in polypeptide, So signal peptide (secretion targeting sequencing) DNA is operably coupled to polypeptid DNA exactly；If promoter controls transcribing of sequence, So it is to be operably coupled to coded sequence；If ribosome binding site is placed in when can make its position translated, then It is to be operably coupled to coded sequence.Typically, " being operably coupled to " means adjacent, then anticipates for secretion targeting sequencing Taste in reading frame adjacent.

In the present invention, term " host cell " is eukaryotic cell.Conventional eukaryotic host cell includes yeast cells, plan South mustard cell, tobacco cell and other plant cell.

Can also be used with Northern engram technology or the table of fluorescent quantitative PCR technique analysis Herba Capsellae peroxidase gene product Reach, i.e. analyze RNA transcript presence or absence in cell and the quantity of Herba Capsellae peroxidase.

The Northern engram analysis of Herba Capsellae peroxidase gene or quantitative fluorescent PCR analysis and Herba Capsellae peroxidase The western blot analysis of specific antibody can be used in combination, to confirm the expression in biological specimen of the Herba Capsellae peroxidase.

Additionally, can be used as the nucleic acid molecules of probe in the present invention, this molecule is generally of Herba Capsellae peroxidase nucleotide 8-100 continuous nucleotide of coded sequence, preferably has 15-50 continuous nucleotide.This probe can be used for detecting sample In whether exist coding Herba Capsellae peroxidase nucleic acid molecules.

The present invention relates to detect the method that whether there is Herba Capsellae peroxidase nucleotide sequence in sample, it includes using The probe stated hybridizes with sample, and then whether detection probe there occurs combination.It is preferred that after this sample is PCR amplification Product, wherein pcr amplification primer thing corresponds to Herba Capsellae peroxidase nucleotide coding sequence, and can be located at the two of this coded sequence Side or centre.Primer length is generally 15-50 nucleotide.

Additionally, according to the Herba Capsellae peroxidase nucleotide sequence of the present invention and aminoacid sequence, can be in nucleic acid homology Property or marking protein homology basis on, screening Herba Capsellae peroxidase homologous genes or homologous protein.

In order to obtain the dot matrix of the Herba Capsellae cDNAs relevant to Herba Capsellae peroxidase gene, shepherd's purse can be screened with DNA probe Dish genomic library, these probes are under low stringent condition, use³²P completely or partially radiates Herba Capsellae peroxidase Obtained by activity mark.The DNA library being best suited for screening is the genomic library from Herba Capsellae blade.Build from interested Cell or the method for genomic library of tissue be that biology field is well-known.It addition, it is many such CDNA library can also buy, such as purchased from Clontech, and Stratagene, Palo Alto, Cal..This screening technique Can identify and the nucleotide sequence of Herba Capsellae peroxidase gene family homology.

The Herba Capsellae peroxidase gene full length sequence of the present invention or its fragment generally can use PCR TRAP, recombination method Or the method for synthetic obtains.For PCR TRAP, can be according to relevant nucleotide sequence disclosed in this invention, especially Some conserved sequences design primer, and with commercially available genomic library or made by conventional method well known by persons skilled in the art Standby genomic library, as template, expands and obtains relevant sequence.When sequence is longer, it is often necessary to carry out twice or repeatedly PCR Amplification, is stitched together the fragment amplified by proper order the most again.

Once obtain relevant sequence, it is possible to obtain relevant sequence in large quantity with recombination method.This is typically will It is cloned into carrier, then proceeds to cell, then by conventional method relevant sequence of isolated from the host cell after propagation.

Additionally, can also be used with the method for artificial chemistry synthesis to synthesize relevant sequence.Before the application, prior art is the completeest Entirely can be attached the most again by first synthesizing multiple polynucleotide small fragment and obtain code book invention Herba Capsellae peroxide The nucleotide sequence of enzyme.Then, this nucleotide sequence can be introduced various existing DNA molecules (such as carrier) and cell in this area In.Additionally, sudden change is introduced in protein sequence of the present invention also by chemosynthesis.

In addition to producing with recombination method, the fragment of albumen of the present invention can also be used with solid phase technique, by being directly synthesized peptide Produced (Guan X, Chaffey PK, Zeng C, Tan Z. New methods for chemical protein Synthesis. Top Curr Chem. 2015,363:155-92).Synthetic protein can by hand or automatically in vitro Carry out.For example, it is possible to the 431A type peptide synthesizer (Foster City, CA) of Applied Biosystems from dynamic circuit connector Become peptide.Each fragment of chemosynthesis albumen of the present invention can be distinguished, the most chemically connected to produce dividing of total length Son.

The Herba Capsellae peroxidase of the present invention can improve the cold tolerance of industrial crops by transgenic technology, in the whole world In the case of soil Population explosion the most in short supply, the present invention has great application prospect.

Accompanying drawing explanation

Fig. 1 is Herba Capsellae peroxidase and the systematic evolution tree of the peroxidase protein family member in other species divides Analysis result.

Fig. 2 is Cold exposed (A), cold-induced (B), Herba Capsellae peroxidating under various hormones (C) and various ion (D) treatment conditions Thing enzyme gene expression analysis in Herba Capsellae leaf, root, stem.

Fig. 3 is for turning hygromycin gene PCR qualification result in Herba Capsellae peroxidase gene Nicotiana tabacum L. resistance Seedling.Swimming lane 1. DNA marker；2. wild-type tobacco；3,4 blanks；5. turn empty carrier Nicotiana tabacum L.；6. Agrobacterium positive bacteria；7-13. turn Genetic tobacco resistance Seedling.

Fig. 4 is for turning genes of interest PCR qualification result in Herba Capsellae peroxidase gene Nicotiana tabacum L. positive Seedling.Swimming lane 1. DNA marker；2-5 transgene tobacco positive Seedling.

Fig. 5 is that in transgene tobacco positive plant, Herba Capsellae peroxidase gene expression measures (A), cold coercing down turns base Because of the Phenotypic Observation (B) of Nicotiana tabacum L. positive plant and the measurement result (C) of the cold-resistant physical signs of transgene tobacco Seedling plant.

Fig. 6 is the qualification result of active oxygen in transgene tobacco positive plant.

Fig. 7 is the expression measurement result of source cold acclimation protein in transgene tobacco positive plant.

Detailed description of the invention

The test data concrete below in conjunction with laboratory and in conjunction with specific embodiments, is expanded on further the present invention.These are real Execute example be merely to illustrate the present invention rather than limit the scope of the present invention.The experiment of unreceipted actual conditions in the following example Method, generally according to described in normal condition condition (such as Pehanorm Brooker (work), Huang Peitang (main translate). molecular cloning is tested Guide (fine works version), Chemical Industry Press, 2008) or according to the condition proposed by manufacturer.

The separation of embodiment 1 Herba Capsellae peroxidase gene and qualification

1. the cultivation of Herba Capsellae seedling

Herba Capsellae seed source, is seeded in containing MS in Shanghai City seeds company, Herba Capsellae seed after 75% ethanol disinfection processes₀ In culture medium, seedling is transplanted to after being placed at 26 DEG C cultivation 2w in soil (Vermiculitum: black earth: perlite=9:3:1) continue life Long, in growth course, periodicity of illumination is 16h illumination, and 8h is dark, and humidity keeps constant moderate.

2. the extracting and the extraction of RNA of Herba Capsellae leaves genomic DNA

Take appropriately sized Herba Capsellae blade, pulverize in liquid nitrogen, be respectively adopted plant RNA extraction test kit (CW0588) (Beijing CoWin Bioscience Co., Ltd.) and CTAB method extract total serum IgE and the complete genome DNA of Herba Capsellae.Agarose gel Electrophoresis detects the quality of RNA and DNA respectively.

3. the clone of Herba Capsellae peroxidase gene coding region core sequence

According to arabidopsis (GenBank Accession No.:NP_172018.1) and Caulis et Folium Brassicae capitatae (GenBank Accession No.:XP_013600777.1) the peroxidase 3(peroxidase 3 in) conserved sequence of gene coding region, designs a pair Primer CbRCIF2(5-CTGAAGGACCTTGTCTTACTCTCC-3(is designated as SEQ ID NO.5) and CbRCIR(5- TTAACTATTTGCAACGGAACATTGCCT-3(SEQ ID No.4)), utilize RT-PCR technology amplification to obtain coding region Sequence, the PCR primer that amplification obtains detects through the agarose gel electrophoresis of 1.0%, obtains a strongest fragment of specificity, long Degree is about 250bp.Reclaim this fragment and be connected on pMD-19T carrier, product will be connected and convertE. coil DH5a experiences State cell, it is 267bp that order-checking obtains sequence length.Analyze with Blast and find, the nucleotide sequence of this fragment and arabidopsis (A. thaliana) RCI3A(ATU97648) mRNA sequence homology be 94%, with Caulis et Folium Brassicae capitatae (Brassica oleracea) The homology of the mRNA sequence of XM_013728218.1 is 92%, it was demonstrated that the correctness of this sequence.

4. the clone of Herba Capsellae peroxidase gene sequence

It is divided into three phases to carry out:

(1) clone of intermediate sequence uses step 3 synthetic primer, with genomic dna sequence for template is, carries out PCR amplification. PCR reaction system is: 10 × PCR Buffer 5 μ L；MgCl₂(25mM) 10μL；dNTP Mix (10mM) 5μL；Taq Archaeal dna polymerase (5U/ μ L) 1 μ L；Primer1 (10 μM) 1μL；Primer2 (10 μM) 1μL；DNA1 μL；dH₂O 26 μL;Cumulative volume 50 μ L.Use program is: and 94 DEG C of for 5 min, 30 cycles (94 DEG C of for 30 sec, 56 DEG C for 30 sec, 72℃ for 50 sec).The PCR primer that amplification is arrived detects through the agarose gel electrophoresis of 1.0%, obtains one The fragment that bar specificity is the strongest, a length of about 450bp.Reclaim this fragment and be connected on pMD-18T carrier, product will be connected ConvertE. coil DH5a competent cell, it is 447bp that order-checking obtains sequence length.With step 3 sequence alignment analysis, find In addition to the one section of intron sequences having more, remaining sequence is the most identical with step 3, illustrates that the sequence that we obtain is Herba Capsellae peroxide Compound enzyme genome intermediate sequence；

(2) sequence obtained according to step (1) separately designs the primer of amplification intermediate sequence upstream, and two of which forward primer divides It is not designated as CbRCI5-1(5-GAGATACACCGATAGTGTGAGCCCCGGA-3(and is designated as SEQ ID No.6)) and CbRCI5-2 (5-TGTTTTGCGGCTCCCTGGATCCATCTCA-3(is designated as SEQ ID No.7)).The Herba Capsellae genome extracted with step 2 DNA is template, utilizes the Universal GenomeWalker Kit(Clontech that CIONTECH company provides) in test kit Adapter-primer AP1 and AP2 carry out nested amplification respectively with upstream gene Specific primer pair, gained purpose band i.e. includes To 5 ' the outer sequences having moved certain distance of end of intermediate sequence.The reaction system of PCR and program are all entered by the description of test kit OK.The amplification of upstream sequence detects through the agarose gel electrophoresis of 1.0%, obtains a strongest fragment of specificity, a length of About 1200bp.Recovery is separately recovered the two fragment and is connected on pMD-19T carrier, will connect product and convertE. coil DH5a competent cell, order-checking obtains a length of 1140bp of upstream sequence；

(3) sequence assembly is carried out according to step (1) and (2) amplified fragments, then according to splicing sequential design amplification total length primer, Primer sequence respectively CbRCIF(5-AACCCCCACAACTTTAAAGATGAAC-3(is designated as SEQ ID No.8)), described downstream Primer is CbRCIR(5-TTAACTATTTGCAACGGAACATTGCCT-3(SEQ ID No.4)).The Herba Capsellae extracted with step 2 Genomic DNA is that template carries out PCR amplification acquisition full length sequence.PCR reaction system is identical with step (1) with program, and amplification is arrived PCR primer through 1.0% agarose gel electrophoresis detect, obtain a strongest fragment of specificity, a length of 1500bp is left Right.Reclaim this fragment and be connected on pMD-19T carrier, product will be connected and convertE. coil DH5a competent cell, order-checking Obtaining sequence length is 1532bp.Identical with the sequence that step (1) and step (2) are spliced, illustrate that the sequence that we obtain is shepherd's purse Dish peroxidase gene group complete sequence.

The bioinformatic analysis of embodiment 2 Herba Capsellae peroxidase gene

1. genomic organization

The CDS sequence (AY566573) of the sequence obtained with Herba Capsellae peroxidase gene is compared, finds Herba Capsellae gene Group sequence comprises 3 introns, and 3 introns all comprise gt ag intron conserved sequence, removes intron, Herba Capsellae peroxidating The CDS sequence of thing enzyme gene comprises 9811bp.

2. homology analysis

Use http://www.ncbi.nlm.nih.gov/blast website by Protein-Protein blast search with And other peroxidase protein family member include arabidopsis (A. thalianaPeroxidase (NM100405 in) And NP188814), Semen sojae atricolor (Glycine maxPeroxidase (AAD11481) in), the Cortex Eucommiae (EucommiaulmoidesPeroxidase (AAU04879) in), Herba Spinaciae (SpinaciaoleraceaPeroxidating in) Thing enzyme (CAA76374), Oryza sativa L. (Oryza sativaPeroxidase (CAH69331) in), duckweed (SpirodelapolyrrhizaPeroxidase (CAA80502) in), Nicotiana tabacum L. (NicotianatabacumMistake in) Oxide enzyme (BAA07664), Semen Maydis (Zea maysPeroxidase (AAS75418) in), Brassica campestris L (B.rapaIn) The aminoacid sequence of BrCOR (ABF60663) by Protein-Protein BLAST analyze (http: // Www.ncbi.nlm.nih.gov/blast) and Vector NTI v10.0 software analysis, higher homology is demonstrated, wherein The highest with the homology degree of the peroxidase AtRCI3 of arabidopsis (concordance 95%, similarity 94%).

3. phylogenetic analysis

Application Clustal X software, by ortho position phase connection, builds the evolution of CbRCI3 and other peroxiredoxins member Tree (Fig. 1).From protein sequence comparison result and evolutionary relationship, CbRCI3 Yu AtRCI3 closest to.

The expression specificity analysis of embodiment 3 Herba Capsellae peroxidase

1. the various treatment conditions of Herba Capsellae plant

(1) cold-induced process: by growing the Herba Capsellae seedling of 4w under 22 DEG C of long-day conditions, move to 4 DEG C and process 4h, 8h respectively, 24h；

(2) Cold exposed processes: will grow the Herba Capsellae seedling of 4w under 22 DEG C of long-day conditions, is sequentially placed into 12 DEG C and processes 4d, at 4 DEG C Reason 4d, 0 DEG C processes 2h；

(3) HORMONE TREATMENT: will grow the Herba Capsellae seedling of 4w under 22 DEG C of long-day conditions, the most soaking at 5 μMs of GA, 300 μMs MeJA, 20 μMs of IAA, 100 μMs of ABA aqueous solutions process 1h, 6h, 24h；

(4) solion processes: will grow the Herba Capsellae of 4w under 22 DEG C of long-day conditions, uses 80mM KCl, 50mM respectively MgCl₂, 5mM ZnCl₂, 30mM LiCl, 0.1mM CuCl₂, 80mM CaCl₂Aqueous solution soaking processes 4h, 8h, 24h.

2. Herba Capsellae leaf, root, the Total RNAs extraction of three kinds of stem tissue

Plant RNA extraction test kit (CW0588) (Beijing CoWin Bioscience Co., Ltd.) is used to extract Herba Capsellae respectively The total serum IgE of root, stem, three kinds of leaf tissue, step and embodiment 1(2) identical.

3. quantitative fluorescent PCR analysis

Fluorescent quantitative PCR technique is used to detect the change of Herba Capsellae peroxidase gene expression under various treatment conditions, experiment Three groups of repetitions are set, with Herba Capsellae 18s rRNA gene (GenBank Accession No.:AY662285) as internal reference.Result shows Show that the background expression of Herba Capsellae peroxidase gene only has in relatively low expression, stem and leaf almost without expression in root.Cold lure The expression in each tissue of the Herba Capsellae peroxidase gene all can be increased substantially after leading (Fig. 2 A) and Cold exposed (Fig. 2 B) process Amount.It is different that hormon processes the expression regulation result to Herba Capsellae peroxidase.ABA can be remarkably reinforced Herba Capsellae peroxidase The expression of gene, and increase over time, expression can persistently rise；MeJA can instantaneous raising Herba Capsellae peroxidase base Because of expression, present feedback suppression；GA₃The expression of Herba Capsellae peroxidase gene can be suppressed；IAA to the expression of its gene without shadow Ring (Fig. 2 C).Metal ion treatment finds, calcium ion, copper ion and lithium ion all can induce the table of Herba Capsellae peroxidase gene Reaching, magnesium ion, zinc ion, potassium ion then can suppress the expression (Fig. 2 D) of Herba Capsellae peroxidase gene.

The leaf disk method Plant Transformation that embodiment 4 is agriculture bacillus mediated

1. the structure of plant expression vector

In the present embodiment, after Herba Capsellae peroxidase gene is placed in 35S promoter, build a plant expression vector, For Plant Transformation.

2. the agrobacterium-mediated transformation conversion to Nicotiana tabacum L.

(1) cultivation of Agrobacterium.Picking list bacterium colony, adds in 2mL Agrobacterium fluid medium, 28 DEG C of overnight incubation；Take 2mL with Upper culture, adds in 50mL Agrobacterium fluid medium, cultivates to OD for 28 DEG C₆₀₀=0.6-1.0；8000rpm is centrifuged 10min, Collect thalline, use MS₀Resuspended, make OD₆₀₀=1.0；

(2) co-culture.Take children's blade tender, healthy and strong of Nicotiana tabacum L. aseptic seedling, remove master pulse, blade is cut into about 0.8cm × 0.8cm Leaf dish, is placed on MS₁In culture medium, anti-uppermost leaf dish is dehydrated；Leaf dish is put in Agrobacterium culture fluid, 190rpm, on 28 DEG C of shaking tables Contaminate 10min；Leaf dish pulled out by medication spoon, is placed in the absorbent paper of sterilizing, blots the unnecessary bacterium solution of Ye Panshang；The leaf dish that will blot Lie in the MS adding a cover a metafiltration paper₁In culture medium, about 25 DEG C, co-culture about 2d in dark place；

(3) induced bundle is sprouted.After co-culturing, according to the following steps the Agrobacterium of leaf panel surface is washed off, the most frequently shake, make leaf Dish is fully contacted following solutions: sterilized water, 15min；Sterilized water+Carbenicillin (350mg/L), 15min；MS₀+ carboxylic benzyl penicillium sp Element (350mg/L), 20min.Leaf dish pulled out by medication spoon, is placed in absorbent paper, blots excessive moisture；Leaf dish is placed on MS₂Cultivate On base, leaf plate edge is gently pressed in culture medium；About 26 DEG C, 16h illumination cultivation；

(4) root induction.At MS₂After inducing about 4w in culture medium, the plumelet grown by leaf margin cuts with leaf dish from base portion, by children Bud cutting enters MS₃Root induction in culture medium, about 26 DEG C, 16h illumination cultivation.

The Molecular Detection of embodiment 5 transgene tobacco

1. the extraction of transgenic tobacco leaf DNA and the PCR of resistant gene detect

The extraction step of transgenic tobacco leaf DNA can refer to embodiment 1(2) method.With the transgene tobacco Seedling leaf extracted Sheet genomic DNA is template, uses hygromycin gene specific primer Hyg-F(5-GTCGAGAAGTTTCTGATCG-3(to be designated as SEQ ID No.9)) and Hyg-R(5-GTTTCCACTATCGGCGAGTACT-3(be designated as SEQ ID No.10)) carry out PCR expansion Increase, with wild-type tobacco and the tobacco sample negative control proceeding to empty carrier, 1% agarose gel electrophoresis detection transgene tobacco Whether containing hygromycin resistant gene (700bp) in genome, result shows at Fig. 3, and testing result shows that having 35 turns Gene resistance Seedling includes hygromycin resistant gene.

2. the PCR detection of genes of interest in transgene tobacco

With extract transgene tobacco genomic DNA as template, use Herba Capsellae peroxidase gene specific primer carry out PCR expands, and primer sequence is: CbRCIF3(5-GGATGCGATGGATCAGTGCTTATA-3(is designated as SEQ ID No.11)) CbRCIR (5-TTAACTATTTGCAACGGAACATTGCCT-3(SEQ ID No.4)).With wild type and the cigarette turning empty carrier Grass sample is negative control, whether contains Herba Capsellae peroxidase purpose in 1% agarose gel detection transgene tobacco genome Gene (1200bp), result shows at Fig. 4, and testing result finds have 30 Seedlings to contain Herba Capsellae peroxidating in 35 Nicotiana tabacum L. resistance Seedlings Thing enzyme gene.

3. the RNA of transgene tobacco extracts and the qualification of destination gene expression amount

The extraction of transgene tobacco RNA can refer to embodiment 1(2) method.In fluorescence quantitative PCR detection 30 strain transgene tobacco The expression of Herba Capsellae peroxide gene, the primer sequence of use is: Realtime-CbRCI35-F (5- CATTAGCCAACATTCCTCCTCCGACCA-3(is designated as SEQ ID No.12)) and Realtime-CbRCI35-R (5- CTGACTGAAACAGACCTCTACGCTTG-3(is designated as SEQ ID No.13)), with the GAPD gene (GenBank in Nicotiana tabacum L. Accession No.:AJ133422) it is internal reference.Found that relative to wild-type tobacco, its expression of different transgenic lines Amount difference, in transgene tobacco, gene expression amount has raised, and Fig. 5 A is shown that wherein 2 strain transgene tobacco positive plant Expression.

The freezing test of embodiment 6 transgene tobacco positive plant

Choose 2 strains that expression is higher, after continuing plantation, obtain pure lines.Two described transgenic pure lines plant are placed in 12 DEG C of illumination cultivation, cold adaptation 4d, it is sequentially placed into 4 DEG C afterwards and processes 24h ,-4 DEG C process 1h, recover 3d at 22 DEG C, shoot record Transgene tobacco positive Seedling and non-transgenic wild-type tobacco Seedling and proceed to the phenotypic difference (Fig. 5 B) of empty carrier tobacco seedling.Result Show that under 22 DEG C of normal temperatures, transgene tobacco is without the phenomenon such as growth delay, dwarfing；Non-transgenic and turn zero load under 4 DEG C of cold treatments Body matched group plant is damaged to plants caused by sudden drop in temperature more serious, leaf withering, and stem lodges, and transgene tobacco positive Seedling is damaged to plants caused by sudden drop in temperature slightly, only minority Lobar atrophy；At-4 DEG C, non-transgenic reference group plant is damaged to plants caused by sudden drop in temperature seriously, and leaf withering aggravates with the phenomenon of stem lodging, transgenic cigarette Grass is damaged to plants caused by sudden drop in temperature equally, leaf withering occurs, but phenomenon is lighter.Transgene tobacco positive Seedling and comparison are placed back in 22 DEG C and recovers raw Long, after 3d, transgene tobacco positive Seedling can return to normal growth state, and matched group tobacco seedling then can atrophy, death.

The mensuration of the cold-resistant physical signs of embodiment 7 transgenic positive Nicotiana tabacum L.

Take the tobacco leaf after embodiment 6 being processed and measure blade electrical conductivity, relative water content and glucose content.Setup Experiments Three groups of repetitions, by t-test evaluation transgenic group and the significant difference of matched group.

1. the mensuration of blade electrical conductivity

Use 8mm bore card punch at 8, the master pulse two side draw leaf dish of each tobacco plant same position blade, be dipped in 10ml Evacuation 2h in Milli-Q water, uses conductivity meter DDS-11A(Shanghai Suo Shen) detection bulk electrical conductivity.100 DEG C are boiled rear cold But to room temperature, absolute electrical conductivity is detected.Relative conductivity (%)=bulk electrical conductivity/absolute electrical conductivity × 100%.Result shows Cold coercing lower matched group tobacco leaf and coerce lower electrical conductivity significantly increase cold, membrane permeability dramatically increases；And transgene tobacco Positive plant leaf membrane permeability increases the slowest, and amplitude is the least, illustrates that its cytoplasma membrane extent of damage is lighter.Statistics Both significant differences (Fig. 5 C) of display.

2. relative to Water content determination

Use 6 mm bore card punch at 8, the master pulse two side draw leaf dish of the various tobacco plant processed same positions blade, Weigh fresh weight (FW)；Leaf dish is put into evacuation 4 h in Milli-Q water, weighs weight in wet base (TW)；Leaf dish absorbent paper is blotted, 85 DEG C are dried 24 h, weigh dry weight (DW)；Water content (%=(FW-DW)/(TW-DW) × 100% relatively.Result shows cold coercing Under, the relative water content of cold quick plant tobacco is remarkably decreased, and shows that moisture coerces lower scattering and disappearing with cytoactive composition cold.Compare Wild type and empty vector control, two high expressed strains of transgene tobacco significantly higher relative to water content (Fig. 5 C).Meanwhile, different Relative water content value no significant difference between transgenic line, overexpression can be complete in plant to show Herba Capsellae peroxidase Become the low-temperature protection of cell.

3. glucose content measures

Use glucose content (HK method) to measure test kit (Sigma-Aldrich, Inc., USA) to be used for detecting tobacco leaf phase Content to glucose.Each processing method every kind processes under gradient, and each strain takes the blade of 4 strain plants, and every strain takes identical The position about 1.5 square blade of cm, claims leaf quality, adds 1.5ml deionized water, and 65 DEG C process 7 h, then evacuation 2 H, obtains the glucose extracting solution of sample.Measure NADH at 340 nm with spectrophotometer (Eppendorf, Germany) to produce The light absorption value of thing, deionized water is comparison.Glucose content is directly proportional to absorbance, and computing formula is: glucose content= 0.319×[Atest-(Asample blank +Areagent blank)]。A _Test, A _{Sample Blank}And A_{Reagent Blank} Be respectively sample cell, the Glucose Assay Reagent equal-volume deionized water in sample cell (is replaced by sample blank pipe Generation), the absorbance of reagent blank pipe (changing the sample glucose extracting solution in sample cell into equal-volume deionized water).Fructus Vitis viniferae Sugar plays a role in the osmotic potential of regulation cell, surveys from the glucose content of WT lines with transgenic positive plant leaf Determine result it can be seen that turn the glucose inside cells Content glucose content (figure higher than wild type of Nicotiana tabacum L. positive plant 5C), show that glucose inside cells content improves the hydropenia and cell injury caused with response low temperature when being coerced by Chilling stress.

The peroxide analysis of the plant of embodiment 8 transgene tobacco

The peroxide analysis of transgene tobacco uses DCFH-DA fluorescent probe, and this probe may pass through cell membrane thus by peroxide Compound enzyme enzymolysis becomes DCFH, stays intracellular.The most intracellular DCFH can be aoxidized by active oxygen and send fluorescence.Experiment knot Shu Shi, first cuts leaf tissue, is then immersed in the DCFH-DA fluorescent probe solution that concentration is 10 μMs, incubates under the conditions of 37 DEG C Educate 20min, use deionized water rinsing blade surface afterwards, make the DCFH-DA of blade cell surface attachment be cleaned out, in order to avoid impact Follow-up observation, interferes.Result display transgene tobacco positive plant is compared to wild-type tobacco adjoining tree peroxidating Thing showed increased (Fig. 6), illustrates that Herba Capsellae peroxidase is a kind of peroxidase that can produce ROS.

The Molecular mechanism analysis on bio that the cold resistance of the plant of embodiment 9 transgene tobacco is formed

Be can be seen that the cold tolerance of transgene tobacco positive plant has had by the phenotype of above Cold exposed to carry largely Height, the raising of this ability inevitable with plant cell membrane protected protein etc. material relevant, say, that the transgene tobacco positive is planted Herba Capsellae peroxidase in strain may improve the cold resistance of Nicotiana tabacum L. by activating the key gene of the signal pathway of cold response. Nicotiana tabacum L. exists the cold response approach related gene similar to arabidopsis, whereinNtDREB1WithNtDREB3CodingCBF/DREBClass transcription factor, plays an important role in cold signal pathway；NtERD10aWithNtERD10bThen Some of coding downstreamCORGene；NtTSI1It is a transcription factor relevant with disease etc..Transgene tobacco RNA's Extraction can refer to embodiment 1(2) method.With 5 endogenous cold responses in fluorescent quantitative PCR technique detection transgenic tobacco plant The expression change of gene.Result shows, under 26 DEG C of condition of culture, relative to matched group Nicotiana tabacum L., described transgene tobacco sun The expression of the cold-induced approach related gene of property plant self has had a certain amount of rise；After processing at 4 DEG C, transgenic In Nicotiana tabacum L. positive plantNtDREB1,NtDREB3,NtERD10a, andNtERD10bExpression Raise and be also significantly stronger than adjoining tree (Fig. 7).NtTSI1Expression nothing in transgene tobacco and check plant is the poorest Not.Thus illustrating, Herba Capsellae peroxidase gene can participate in the signaling pathways in cold tolerance approach, transmits through signal The downstream cold inducible gene expression amount making Nicotiana tabacum L. endogenous is greatly improved, thus improves Plant chilling resistance.

<110>Fudan University

<120>Herba Capsellae peroxidase gene and the application in improvement economic plants winter resistance thereof

<130> 001

<160> 13

<170> PatentIn version 3.3

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Claims

1. the Herba Capsellae peroxidase gene with low temperature induction characteristic separated from Herba Capsellae, it is characterised in that described Herba Capsellae peroxidase gene selected from one below:

(1) its nucleotide sequence is as shown in 20-1532 bit base in SEQ ID No.1；

2. a recombiant plasmid, it is characterised in that containing the Herba Capsellae peroxidase gene described in claim 1.

3. a Herba Capsellae peroxidase, it is characterised in that described Herba Capsellae peroxidase is selected from one below:

(1) its aminoacid sequence is as shown in SEQ ID No.2；Or

(2) there is conservative variation's polypeptide or its active fragment of the polypeptide of SEQ ID No.2 aminoacid sequence, or its activity Derivant.

4. the preparation method of the Herba Capsellae peroxidase gene described in a claim 1, it is characterised in that comprise the following steps:

(3) genomic walking technology clone is used to obtain Herba Capsellae peroxidase gene full-length genome sequence, wherein, use Forward primer is: SEQ ID No.3, and downstream primer is SEQ ID No.4.

5. the preparation method of the recombiant plasmid described in a claim 2, it is characterised in that comprise the following steps:

(1) design primer pair, amplifies the intact DNA fragments of Herba Capsellae peroxidase gene described in claim 1, expands institute With primer to for: forward primer is SEQ ID No.3, and downstream primer is SEQ ID No.4；

(2) described intact DNA fragments is cloned into intermediate carrier pMD18-T, is further cloned into plant expression vector p1304 On, obtain described recombiant plasmid.

Preparation method the most according to claim 5, it is characterised in that also include introducing Restriction Enzyme to described forward primer Cut siteNcoI, introduces restriction enzyme site to described downstream primerBstEII。

7. the Herba Capsellae peroxide described in claim 1 protects the recombiant plasmid described in volume thing enzyme gene or claim 2 at plant cold resistance Application in property and tolerance to cold.

Application the most according to claim 7, it is characterised in that described plant is the crop with economic worth, including water Rice, Semen Tritici aestivi, Semen Maydis, Cotton Gossypii, Brassica campestris L, Fructus Lycopersici esculenti and Fructus Cucumidis sativi.

Application the most according to claim 7, it is characterised in that described recombiant plasmid is proceeded to Agrobacterium tumefaciems EHA105 In, it is subsequently used for converting purpose plant, improves plant to cold toleration.

Application the most according to claim 7, it is characterised in that utilize transgenic technology that coding has Herba Capsellae peroxidating The nucleotide sequence of thing peptide activity is transformed into plant, and to improve the plant toleration to cold, its step is as follows:

(1) nucleotide sequence of the purification that coding has Herba Capsellae peroxidase activity polypeptide is operably coupled to plant and expresses After regulating and controlling sequence, form the plant expression vector containing Herba Capsellae peroxidase gene, described nucleotide sequence and SEQ ID No. nucleotides sequence from nucleotide 20-1532 position shows the homology of at least 70% in 1；

(2) expression vector in step (1) is proceeded to Agrobacterium, the Agrobacterium containing expression vector is trained altogether with eukaryotic host cell Support, under the conditions of 22-28 DEG C, after light culture 1-2d, by screening, it is thus achieved that the conversion containing Herba Capsellae peroxidase gene is thin Born of the same parents finally regeneration of transgenic plant and offspring thereof, including plant seed and plant tissue.